Apparatus for separating proteins from blood plasma

ABSTRACT

An apparatus is provided for reacting a solution of a precipitating medium of alkaline metal chlorides at a fixed rate of delivery with plasma continuously separated from blood, filtering the precipitate formed by the reaction, removing the solution of precipitating medium from the plasma following the use of the medium, adjusting the purified treated plasma, and returning the purified plasma to the blood in an amount equivalent to that of the continuously separated plasma. To enhance the precipitative separating action of alkaline metal chloride on plasma protein without detracting from the advantages of the alkaline metal chloride, use is made of a solution of precipitating medium comprising a mixture of the alkaline metal chloride and an amino acid, the latter being added in an amount sufficient to promote the precipitating effect of the alkaline metal chloride.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an agent for the separation and removal ofproteins from blood plasma by the salting-out effect, and to anapparatus which utilizes the agent to separate and remove specificproteins such as immune complexes, immunoglobulins, fibrinogen and othersoluble macromolecular proteins from blood plasma. The apparatus is usedin removing pathogenic substances from a large quantity of collectedblood followed by reinfusion of the blood, and in the purification ofplasma by removal of pathogenic substances from a large quantity of poolblood.

2. Description of the Prior Art

It is becoming clear that immunoglobulins, immune complexes, complement,fibrinogen and other soluble macromolecular substances contained inblood play a role in causing autoimmune diseases, rheumatoid arthritisand other antigenic diseases. These disease treated by a plasma exchangeprocess which includes removing plasma containing the harmful substancesfrom the patient's blood and replacing the plasma with a substitutefluid. This process was first used in 1963 for the treatment ofmacroglobulinemia and since then has been tried in the treatment of manyillnesses. With the development in recent years of membrane-type plasmaseparators, plasma exchange has become simpler and, hence, more widelypracticed. However, the wider application of this treatment has beenaccompanied by increased consumption of the substitute fluids (e.g.,FFP, agents made of albumin and the like) the supply of which has, as aresult, become limited and higher in cost. An additional problem is thatthe infusion of a large quantity of human plasma ca bring aboutunwelcome side effects such as hepatitis, alergic reactions and serumsickness.

A method now undergoing research for dealing with the foregoing problemsis so-called plasma purification or cleansing which, rather than relyingupon a substitute fluid, selectively removes the macromolecular proteinscausing the particular illness from the patient's blood plasma andreinfuses the patient with his or her own cleansed blood plasmacontaining albumin and other useful plasma ingredients. Plasmapurification processes known so far include a process for removingmacromolecular protein from blood plasma by using a membrane having apore diameter smaller than that of a plasma separating membrane, and aprocess for the adsoptive removal of macromolecular protein from bloodplasma by relying upon an adsorbent. While the former has tentativelyshown some efficacy in clinical use and has won some standing as acurative means, the latter is still in the basic research stage and onlypartial clinical use has been reported. Both processes have drawbacks inthat the former exhibits insufficient selectivity in terms of removingspecific proteins and the latter is incapable of treating a largequantity of plasma at one time. Thus, neither process is truly adequate.Accordingly, there is a need to develop a more effective and efficientapparatus for purifying blood plasma.

In view of these circumstances, the applicant has filed patentapplications for inventions the gist of which is to use a chloride of analkaline metal, e.g., sodium chloride, as an agent, or precipitatingmedium, for separating proteins from plasma by the salting-out effect.The applications filed are Japanese patent application Nos. 58-207463and 58-207464. The proposed separating agent, owing to its weaksalting-out action and low solubility in blood plasma, is effective incausing the specific precipitation solely of macromolecular proteinssuch as fibrinogen and immuno-globulins without resulting in theprecipitation of such useful proteins of low molecular weight asalbumin, even if the rate at which the agent is added to plasma isgreater than that required for saturation.

SUMMARY OF THE INVENTION

An object of the present invention is to solve or mitigate theaforementioned problems involved in supplying a substitute fluid inplasma exchange treatment, and to facilitate the implementation of theplasma exchange treatment, by providing an apparatus for the separationand removal of proteins from blood plasma, which apparatus is improvedin terms of structure and function to fractionate various proteins fromblood plasma selectively and in large quantities by utilizing adifference in solubility.

Another object of the present invention is to provide an agent for theprecipitative separation of proteins from blood plasma, which agentretains the advantages of the abovementioned alkaline metal chloridewhile exhibiting an improved separating action against plasma proteins.

Still another object of the present invention is to provide an apparatuswhich uses the aforementioned plasma protein separating agent toseparate and remove proteins from blood plasma.

According to the present invention, the first-mentioned object isattained by providing an apparatus for the separation and removal ofproteins from blood plasma, which apparatus includes a plasma pump fordelivering plasma from a living body and for returning treated plasma tothe living body in an amount equivalent to that of the plasma delivered,a vessel for accommodating a solution of a precipitating medium forcausing precipitation of a predetermined protein constituent in theplasma, a precipitating medium solution pump operatively associated withthe plasma pump for delivering the solution of precipitating medium at afixed delivery rate, a mixer for mixing the delivered plasma and thedelivered solution of precipitating medium, whereby a precipitate isformed in the mixer, a plasma filter for removing the precipitate formedin the mixer, thereby resulting in filtered plasma, a plasmaconstitutent adjusting unit for removing the precipitating medium fromthe filtered plasma and for producing the abovementioned treated plasmaby adjusting water content and electrolyte, and a control unit fordriving and controlling the plasma pump and the precipitating mediumsolution pump.

According to an embodiment of the present invention, the plasmaconstituent adjusting unit comprises a further plasma pump operativelyassociated with the plasma pump for operating at a flow rate equal to orless than the delivery flow rate of the plasma pump, plasmaconcentrating means operatively associated with the further plasma pumpfor removing water from the plasma in an amount equal to or more thanthat of the added solution of precipitating medium, thereby resulting inconcentrated plasma, and precipitating medium removing means forremoving the precipitating medium from the concentrated plasma and foradjusting the electrolyte of the concentrated plasma.

According to embodiments of the present invention, the precipitatingmedium solution is a solution of various inorganic salts, a solution ofvarious organic solvents, or a solution of an inorganic or organic acid.

It should be noted that the plasma proteins mentioned herein refer toproteins present in the liquid fraction that results when tangibleconstituents, namely blood cells (red blood cells, white blood cells andplatelets) are removed from blood. Though these plasma proteins may bebroadly classified into albumin, globulins (α₁, α₂, β, γ) andfibrinogen, a further subdivision into some 80 types now known can bemade by a more detailed analysis, these proteins including transferrin,haptoglobin, hemopexin, glocoprotein, riboprotein, immunoprotein,compliment and immune complex enzyme. Furthermore, plasma proteins canalso be broadly classified into proteins of high and low molecularweight. The former, namely macromolecular protein, refers toimmunoglobulins, immune complexes, compliment, fibrinogen and the like,while the latter refers to albumin, etc.

The plasma protein separating agent and apparatus of the presentinvention are used for the extraction mainly of macromolecular proteins.

According to the present invention, the second-mentioned object isattained by providing an agent for the precipitative separation ofproteins from blood plasma comprising, as an active ingredient, amixture of an alkaline metal salt and an amino acid in an amountsufficient to promote the fractional precipitating effect of thealkaline metal salt with respect to plasma protein, the amino acid beingat least one selected from the group consisting of neutral amino acid,aspartic acid, cystine, N-acetyltryptophan and tyrosine.

Further, according to the present invention, the third-mentioned objectis attained by providing an apparatus for the separation and removal ofproteins from blood plasma including a vessel for accommodating an agentfor the precipitative separation of proteins from blood plasma, theagent comprising, as an active ingredient, a mixture of an alkalinemetal salt and an amino acid in an amount sufficient to promote thefractional precipitating effect of the alkaline metal salt with respectto plasma protein, the amino acid being at least one selected from thegroup consisting of neutral amino acid, aspartic acid, cystine,N-acetyltryptophan and tyrosine, means for introducing plasma into thevessel, means for separating and removing a precipitate formed insidethe vessel by bringing the separating agent and the plasma into contact,and means for removing at least some of the separating agent constituentfrom the plasma component which remains following removal of theprecipitate.

Other features and advantages of the present invention will be apparentfrom the following description taken in conjunction with theaccompanying drawings, in which like reference characters designate thesame or similar parts throughout the figures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating a preferred embodiment of a bloodpurifying apparatus according to the present invention;

FIG. 2 is a block diagram illustrating another preferred embodiment of ablood purifying apparatus according to the present invention;

FIG. 3 is a flowchart illustrating flow rate control performed by acontrol unit in the apparatus of FIG. 2;

FIG. 4 is a block diagram illustrating still another preferredembodiment of a blood purifying apparatus adapted for batch processingaccording to the present invention;

FIG. 5 is a plan view illustrating the blood purifying apparatus of FIG.4 in greater detail; and

FIG. 6 is a sectional view of a connector for aseptically connectingtubes in the blood purifying apparatus of FIG. 5.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Blood plasma contains proteins of a high molecular weight, such asimmune complexes, immunoglobulins and fibrinogen, as well as proteins ofa comparatively low molecular weight, such as albumin. There is a needin self-purifying plasma treatment to selectively remove only theproteins of high molecular weight and leave the plasma with the usefulproteins of low molecular weight, such as albumin. The inventors haveconducted extensive research with regard to media capable ofprecipitating proteins in plasma and as a result have found thatalkaline metal chlorides, particularly sodium chloride, owing to theircomparatively weak salting-out action and low solubility, cause specificprecipitation solely of the macromolecular proteins such as fibrinogenand immunoglobulins with hardly any precipitation of albumin, even ifthe medium, say sodium chloride, is added to the plasma in an amountgreater than that required for saturation. More specifically, theinventors have discovered that if the sodium chloride added forseparating and removing the precipitate formed by mixing theprecipitating medium with plasma is maintained in the plasma at anamount above that needed for saturation, then strict control isunnecessary, and that there is no change in the effectiveness ofprecipitation or in the composition of the precipitate, as well as nochange in the properties of the protein, even at the high level ofsodium chloride addition. Based on the fruits of this research, theinventors have perfected the present apparatus which is capable ofpurifying blood by separating solely the macromolecular proteins from alarge quantity of blood plasma by on-line implementation of the steps ofplasma separation, precipitate formation, removal of the precipitate andrecovery of the processed plasma.

Preferred embodiments of the present invention will now be describedwith reference to the accompanying drawings.

FIG. 1 is a block diagram illustrating a blood purifying systemaccording to an embodiment of the inventive apparatus for the separationand removal of a protein constituent from blood plasma. A blood pump 1delivers blood collected from a patient into a main conduit 101 made of,e.g., vinyl chloride. The main conduit 101 is connected to a plasmaseparator 2 which, by centrifugal- or membrane-type separation,separates the blood entering from the conduit into plasma and blood cellfractions. The separated plasma from the separator 2 is fed into asecondary conduit 102, also made of vinyl chloride, by a doubleroller-type plasma pump 3 which, at the same time, returns processedblood plasma in amount equivalent to the plasma drawn from the patient.A vessel 4 containing a solution of a precipitating medium is connectedto a precipitating medium solution pump 5 operatively associated withthe plasma pump 3 for delivering the solution of precipitating mediumfrom the vessel 4 at a fixed feed ratio. A mixer 6 receives plasmadelivered by the plasma pump 3 and the solution of precipitating mediumdelivered by the pump 5 for mixing the two together. The outlet of themixer 6 is connected to a plasma filtering unit 7 for removing a proteinfraction which has precipitated inside the mixer 6. The outlet of theplasma filtering unit 7 is in turn connected to a plasma constituentadjusting unit 8 for removing the precipitating medium from the filteredplasma and for adjusting water content and electrolyte. The processedplasma following its adjustment in the adjusting unit 8 is fed by thepump 3 into a plasma mixing unit 9, where the plasma is mixed andrejoined with the concentrated blood comprising the blood cellsoriginally separated from the incoming blood by the plasma separator 2.A control unit 10 acts through the blood pump 1, plasma pump 3 andprecipitating medium solution pump 5 to control the amounts of blood,plasma and precipitating medium solution delivered.

With the above-described arrangement, the patient's blood introducedfrom a blood inlet a is cleansed automatically and continuously beforebeing returned to the patient from a blood outlet b. More specifically,blood drawn from the patient is introduced by the blood pump 1 into theplasma separator 1, where the blood is separated into blood cell andplasma fractions. The plasma resulting from the separation is led intothe secondary conduit 102 by the plasma pump 3 and flows into the mixingunit 6. Meanwhile, the solution of precipitating medium in the vessel 4is delivered to the mixing unit 6, in which mixing with the plasma takesplace, by the plasma medium solution pump 5 rotated at a fixed ratio inoperative association with the plasma pump 3. A precipitate (mainlyfibrinogen and globulin fractions) that forms in the mixing unit 6 isfiltered out by the plasma filtering unit 7 for precipitate removal. Theresulting filtered plasma, which still contains the precipitatingmedium, flows from the filtering unit 7 into the plasma constituentadjusting unit 8. Here the processed plasma is submitted to dewatering,removal of the precipitating medium and adjustment of electrolyte beforebeing delivered to the plasma mixing unit 9 by the double roller-typeplasma pump 3 in an amount equivalent to that of the plasma collectedfrom the patient. The processed plasma mixes with the concentrated blooddelivered by the plasma separator 2 and the mixture of processed plasmaand concentrated blood is reinfused into the patient from the bloodoutlet b.

Examples of the solution of the precipitating medium for proteinseparation are solutions of a variety of salts, namely alkaline metalsalts such as sodium chloride, potassium chloride, sodium sulfate,potassium phosphate and sodium citrate, ammonium salts such as ammoniumsulfate, and the like. In particular, when an alkaline metal salt isused, the amount of salt added need not be strictly controlled, for itwill suffice if control is exercised such that the amount of salt addedis maintained above that necessary for causing the macromolecularproteins to precipitate out of the plasma. In addition, though it ispossible to use an organic solvent such as ethanol, an inorganic acidsuch as hydrochloric acid or sulfuric acid or an organic acid such asascorbic acid, there is the possibility of a change in properties of theproteins when these substances are employed. It is therefore necessaryto provide a set-up in which close attention is paid to temperaturemeasurement and control of pH at mixing.

The control unit 10 sets and regulates the rotating ratios of the pumps3 and 5 mainly in dependence upon both the concentration of theprecipitating medium solution and the protein that is desired to beremoved from the plasma and is adapted to control the average flow ratesof these motors so as to mix in an amount of the precipitating mediumsolution prescribed in accordance with the amount of plasma. Forexample, if ammonium sulfate is used as the precipitating mediumsolution, fibrinogen will substantially precipitate at an ammoniumsulfate concentration in plasma of about 10 g/dl, and both fibrinogenand gamma-globulin will substantially precipitate at an ammoniumconcentration in plasma of about 20 g/dl. Therefore, in case of asaturated solution (about 54 g/dl), each of the proteins can be made toprecipitate and separate from the plasma if the settings are such as toadd the ammonium sulfate at rates of about 23 ml and 59 ml,respectively, with respect to 100 ml of plasma. It takes about 10 to 20mS for this chemical reaction. Flow rate control of this kind canreadily be executed under the control of a microprocessor equipped withthe control program of FIG. 3, described later.

A dialyzer used in ordinary dialysis is adopted as the plasmaconstituent adjusting unit 8. Any dialyzer configuration will sufficeproviding that it has a dewatering capability that enables removal ofthe added amount of precipitating medium solution at less than theusable pressures, as well as a dialyzing capability that enables theconcentration of the precipitating medium to be brought below astipulated value.

FIG. 2 is a block diagram showing a blood purifying system according toanother embodiment of the present invention. Portions similar to thoseshown in FIG. 1 are designated by like reference characters and need notbe described again. In this arrangement, the dewatering and dialysisprocesses performed by the plasma constituent adjusting unit 8 of FIG. 1are performed separately at different stages. Specifically, the plasmafiltered by the filtering unit 7 for precipitate removal is fed into aplasma concentrating unit 11 where the plasma is dewatered to a degreeno less than the amount of precipitating medium solution added. Thedewatering process is performed by a plasma pump 13 operativelyassociated with the plasma pump 3 and controlled to provide a flow rateequal to or less than that of the plasma pump 3. Next, the concentratedplasma from the concentrating unit 11 is fed into a precipitating mediumremoval unit 12, where residual precipitating medium is removed andelectrolyte adjusted. The resulting plasma is then delivered to theplasma mixing unit 9 by the double roller-type pump 3 in an amountequivalent to that of the plasma collected. The concentrated blooddelivered by the plasma separator 2 mixes with the plasma in mixing unit9, whence the resulting mixture is reinfused into the patient from theblood outlet b.

FIG. 3 is a flowchart illustrating an embodiment of flow rate controlexecuted by the control unit 10 of FIG. 2. Flow rate control is the samefor FIG. 1 with the exception of the control step involving the plasmapump 13, which is not provided in the apparatus of FIG. 1. The firststep S1 of the flowchart calls for the pump 1 to be driven andcontrolled at an average delivery rate U (e.g., 60 to 100 ml/min) set asthe average flow rate at which blood is to be collected from a patient.The next step S2 calls for the double roller-type pump 3 to be drivenand controlled at an average delivery rate S (e.g., 15 to 40 ml/min)determined in relation to the delivery rate U. Next, at a step S3, arate k at which the solution of precipitating medium is to be deliveredis determined in dependence upon the kind of plasma protein to beprecipitated, and the pump 5 is driven and controlled at an averagedelivery rate W (=k S) determined based on k. This is followed by a stepS4, at which dewatering is performed to a degree no less than the amountof precipitating medium solution added. To this end, the pump 13 isdriven and controlled in operative association with the plasma pump 3and at an average delivery rate M (≦S), namely at a rate equal to orless than at which the plasma pump 3 is driven. It is then decided at astep S5 whether purification has ended. If the decision here isaffirmative, all pumps are stopped; if negative, the control sequence iscontinued from step S1. In order to avoid too much hemolysis,transmembrane pressure (TMP) is controlled so as not to exceed 45 mmHg.

Thus, the invention provides an apparatus capable of highly efficientseparation and removal of macromolecular proteins such asimmunoglobulins and fibrinogen contained in blood plasma. Preferably, analkaline metal chloride such as sodium chloride or potassium chloride isused as the protein precipitating medium. A precipitating medium of thistype will neither alter the effectiveness of precipitation nor changethe properties of the protein in the plasma even if the medium is addedto the plasma in large quantities. This provides a high level ofstability even when large quantities of plasma are submitted to thepurification treatment. In addition, since the precipitating mediumselectively precipitates solely the proteins of large molecular weightsuch as immunoglobulins and fibrinogen but causes almost noprecipitation of the useful proteins of low molecular weight such asalbumin, the useful constituents (albumin, etc.) can be reinfused intothe patient following the self-purification of the patient's blood;hence, a substitute fluid used in plasma exchange treatment isunnecessary. This solves the problems encountered in the prior art,namely the side effects such as hepatitis and allergic reactions, thehigh cost and the scarcity of substitute fluid supplies. Further,according to the present invention, the process for separating andremoving protein consituents from blood plasma can be practiced on-line,thereby shortening overall processing time and reducing the danger ofbacterial infection.

The inventors have conducted extensive research with regard to alkalinemetal chorides such as sodium chloride in an effort to improve theprecipitative separating action thereof with respect to plasma proteins.As the result of this research, the inventors have found that theprecipitative separating action of these chlorides can be greatlyimproved, without detracting from the earlier mentioned advantagesthereof, by blending with an alkaline metal chloride at least one aminoacid selected from the group consisting of neutral amino acid, asparticacid, cystine, N-acetyltryptophan and tyrosine. The inventors have alsofound that these amino acids enhance the plasma protein-separatingaction not only of alkaline metal chlorides but also of other salts ofalkaline metals.

The alkaline metal salt constituting the effective ingredient of theplasma protein-precipitative separating agent of the present inventionincludes chlorides such as sodium chloride, potassium chloride, lithiumchloride, rubidium chloride, cesium chloride and francium chloride, andfurther includes sulfates such as sodium sulfate. Among these alkalinemetal salts, the effect of adding the amino acid is best exhibited forthe chlorides, as will be set forth below. It has been mentioned earlierthat alkaline metal chlorides have the effect of selectivelyprecipitating only harmful macromolecular proteins, especiallyimmunoglobulins, without precipitating useful plasma constituents suchas albumin even when the chlorides are added in an amount in excess ofthat needed for saturation. This is of great convenience in terms ofcontrolling the concentration of the separating agent with respect toplasma. Sodium sulfate, on the other hand, exhibits a strong salting-outeffect and can result in the precipitation of the useful plasmaconstituents such as albumin if mixed with plasma at too high apercentage. This therefore necessitates strict control of the addedsodium sulfate concentration. However, the salting-out effect even ofsodium sulfate can be enhanced by blending it with an amino acid inaccordance with the present invention.

It has been set forth earlier in the specification that harmful proteinsare precipitated out of blood plasma, the precipitate is removed, theplasma protein-separating agent used is subsequently removed from thepurified plasma fraction, and the resulting plasma is returned to thepatient. Therefore, it is preferred that the alkaline metal salt used bereadily removable from the purified plasma fraction and safe as far asthe patient is concerned. Seen in this light, the most desirablealkaline metal salts that can be used are chlorides, especially sodiumchloride and potassium chloride, which exist naturally in the humanbody.

According to the invention, the amino acids blended with theabovementioned alkaline metal salts are neutral amino acids (e.g.,glycine, alanine, valine, leucine, isoleucine), aspartic acid, tyrosine,N-acetyltryptophan and cystine. A mixture of any two or more of thesemay also be used. The neutral amino acids are the most effective amongthese amino acids, with glycine being the most useful.

Enhancement of the plasma protein-separating effect of the alkalinemetal salt is brought about by the addition thereto of theabovementioned amino acid at a proportion of 5 to 50 wt-% with respectto the total weight of the alkaline metal chloride - amino acid mixture.It is preferred that the amino acid be blended with the alkaline metalsalt at a proportion of 10 to 45% with respect to the total weight ofthe alkaline metal chloride - amino acid mixture.

As to the ratio of added separating agent to plasma, the amount ofseparating agent added should be enough for the alkaline metal salt tomanifest its plasma separating effect, namely its selectiveprecipitating effect. More specifically, the amount of separating agentadded is more than that needed to raise the alkaline metal saltconcentration in plasma to saturation.

It is generally considered that salting out occurs due to neutralizationof the electric charges of the colloid in the aqueous solution. Thealkaline metal salt added to the plasma prote in dissociates into ionsin the plasma and selectively neutralizes and precipitates the harmfulmacromolecular proteins, especially the globulins. Though the mechanismthrough which the addition of amino acid enhances the salting-out effectis not yet known in terms of theory, it seems that the amino acid actsin some way upon the plasma protein constituent to thereby raise theeffectiveness at which salting-out takes place.

FIG. 4 is a block diagram illustrating another embodiment of a bloodpurifying system for selectively precipitating and removing, byso-called batch processing, macromolecular proteins from blood plasmathrough use of the plasma protein-separating agent of the presentinvention. Portions similar to those shown in FIGS. 1 and 2 aredesignated by like reference characters and are not described again indetail.

Blood drawn from a patient is fed into the plasma separator 2 throughthe main conduit 101 by the plasma pump 1. The plasma separator 2separates the incoming blood into a blood cell fraction and plasmafraction, with the latter being fed into the secondary conduit 102 bythe plasma pump 3 and thence to a vessel 14, which accommodates theplasma protein-separating agent, namely the precipitating medium, of thepresent invention.

The plasma fraction introduced into the vessel 14 mixes with theseparating agent of the present invention so that the macromolecularproteins in the plasma are precipitated. After being left standing forfrom 1 to 200 hours, the contents of the vessel 14 are fed into aprecipitate separator, e.g., the plasma filter 7, through a conduit 103.The filter 7 separates and removes the precipitate from the plasma,producing a cleansed plasma fraction with is received in a secondaryvessel 15 via a conduit 104. Connected to the secondary vessel 15 viaconduits 105, 106 is means, e.g., a hollow fiber-type dialyzer 16, forremoving the plasma-protein separating agent from the plasma. Thecleansed plasma fraction in the secondary vessel 15 is fed into thedialyzer 16 via the conduit 105 by the pump 17 and then flows back intothe secondary vessel 15 via the conduit 106. This circulation of thecleansed plasma fraction is repeated as often as desired. The cleansedplasma fraction in the dialyzer 16 is dialyzed for removal of theplasma-protein separating agent by a dialyzing solution, such as anyreadily available for use in ordinary artificial dialysis, which ispassed through the interior of the dialyzer 16 via conduits 107, 108. Ifsodium chloride and (or) potassium chloride, which are constituents ofbody fluids, are used as the alkaline metal component in this case,complete removal thereof by dialysis is unnecessary, for it will sufficeif the chlorides are removed to provide a concentration thereofequivalent to that found in the blood of the human body. The secondaryvessel 15 containing the plasma fraction thus cleansed and purified isexchanged with a substitute fluid vessel 18, described below, and isreinfused into the patient through a path identical with that used forinfusing a substitute fluid.

The blood cell fraction separated by the plasma separator 2 is returnedto the patient through a conduit 109. Prior to that, however, the bloodcell fraction is mixed in the mixing unit 9 with a substitute fluid(also referred to in the art as a replacement or make-up fluid), such asphysiological saline or a 5% albumin solution, which is contained in thevessel 18 and introduced into the mixer 9 through a conduit 110 by thepump 3. The reason for introducing the substitute fluid is that purifiedand cleansed plasma, obtained in the manner described above, is not yetready at the initial stage of the plasma purifying operation. Whencleansed and purified plasma is prepared in the above-described fashion,the substitute fluid vessel 18 is replaced by the secondary vessel 15containing the purified plasma and the purified plasma is returned tothe patient through the same path used for infusing the abovementionedsubstitute fluid.

FIG. 5 shows in greater detail the plasma protein-separating apparatusdescribed above with reference to FIG. 4. The apparatus includes aflexible bag 41 serving as vessel for receiving blood plasma. The bag 41comes equipped with a supply of the separating agent of the presentinvention and has a tube L1 serving as a plasma inflow passage and atube L2 serving as a plasma outflow passage. The tip of the tube L1situated inside the bag 41 is closed. Provided in the wall of the tubeL1 at a location distanced slightly from the tip thereof is a notch (notshown). By tearing away the tip of the tube L1 at the notched portionthereof at use, the interior of the tube can be brought intocommunication with the interior of the bag 41. The end of the tube L2outside the bag 41 is closed. Formed in the wall of the tube L2 at alocation distanced slightly from the tip at said end thereof is a notchsimilar to that described above. The tube L2 is connected to a tube L4communicating with a precipitate removal filter 42, described below, bya tube L3 of a larger diameter. The tube L3 is so arranged that thenotched portion of the tube L2 is situated therein. At use, the tubesL2, L4 are communicated by tearing away the notched portion of the tubeL2.

The tube L3 is connected with the filter 42 via tubes L4, L5 in theabove-described manner. The tubes L4, L5 are aseptically interconnectedby a connector C1, described below. The precipitate removal filter 42 isfor the purpose of filtering out a precipitate formed in the bag 41 bycontact between the plasma fraction and the plasma protein-separatingagent of the present invention. The filtered liquid, namely the cleansedplasma, from the filter 42 is collected in a secondary bag 43 via tubeL6.

A tube L14 has one end inserted deeply in the interior of the secondarybag 43. The other end of tube L14 is connected to one end of a hollowfiber-type dialyzer 44. Connected to the other end of the dialyzer 44 isone end of a tube L8, the other end whereof is connected to thesecondary bag 43. The tubes L14 and L8 form a closed circuit. Thefiltered liquid in the secondary bag 43 is circulated through thisclosed circuit in order to be dialyzed to the degree desired. Thedialyzing solution is introduced into the dialyzer 44 from an inlet 44aand flows out of the dialyzer from an outlet 44b.

The end of the tube L1 outside the bag 41 is connected to a tube L9 viaa connector C2, described below. The tube L9 is in turn connected to atube L10 via a connector C3, described below. Plasma outflow tubes L11,L12 of a plasma separating filter 45 merge and are connected to a tubeL10. Blood introduced into the plasma filter 45 from a blood inlet 45athereof is separated by the filter 45 into a plasma fraction and bloodcell fraction. The blood cell fraction exits from a discharge portion45b of the filter 45.

Connected to the tube L5 is a tube L13 for venting internal air or forintroducing ambient air. Connected to one end of the tube L13 is anantibacterial filter F1 which allows the passage of air therethrough butwhich is impermeable to liquid. The filter F1 prevents the intrusion ofbacteria present in the outside air. Arranged in the tube L5 is aflexible tube E1 driven by a pump to induce the outflow of the contentsin bag 41. An antibacterial filter F2, similar to the filter F1, isconnected to the precipitate-removing filter 42 via a tube L15.

Connected to a tube L7 is a priming tube P1 having a filter F3 arrangedin a section thereof. A flexible tube E2 similar to the flexible tube E1is arranged upstream of the tube L7. A pressure monitor M1 is arrangedin a tube L8 to monitor pressure internally of the tube. A priming tubeP2 having a filter F6 disposed in a section thereof is connected to thetube L14. Antibacterial filters F4, F5, similar to those mentionedabove, are connected to the pressure monitor M1. Likewise, a primingtube P3 having a filter F7 arranged in a section thereof is connected tothe tube L9. Arranged upstream of the tube L9 are a flexible tube E3 anda pressure monitor M2.

The connector C1 interconnecting the tubes L4, L5, the connector C2interconnecting the tubes L1, L9, and the connector C3 interconnectingthe tubes L9, L10 are all adapted to make these connections in anaseptic manner. A connector of such type is as disclosed in thespecification of Japanese patent application Laid-Open No,. 57-211353.The disclosed connector comprises first and second connector portions.The first connector portion, in the shape of a short tube of a requisitelength, is made of a heat- and corrosion-resistant material (such as aceramic, stainless steel, titanium) and has a male engagement portionincluding a plug side formed in a male mold. The second connectorportion, also in the shape of a short tube of a requisite length, ismade of a heat- and corrosion-resistant material and has a femaleengagement portion including a plug end formed in a female mold, thefemale engagement portion mating with the male engagement portion of thefirst connector portion. The two connector portions are locked togetherby a locking mechanism which does not rely upon threaded engagement, andthe sides of the connectors that are connected to the respective tubesare each covered by a fitted cylindrical supporting body made of anadiabatic material (such as silicone rubber, Teflon resin or cork). Witha connector of this type, the first and second connector portions arecapable of being connected together while being directly heated byapplication of a flame, thereby assuring sterilization. This makes itpossible to achieve an aseptic connection.

Among the connectors of this type disclosed in the abovementionedspecification of the laid-open application, those illustrated in FIGS. 5and 11 of the specification, and above all in FIG. 11, are particularlywell-suited for use as the connectors C1, C2, C3 of the proteinseparating apparatus of the present invention. One example of theseconnectors will now be described with reference to FIG. 6.

The connector depicted in FIG. 6 comprises a first cylindrical body 51made of a heat- and corrosion resistant material and corresponding tothe abovementioned first connector portion, and a second cylindricalbody 53 made of a heat- and corrosion resistant material andcorresponding to the abovementioned second connector portion. The firstcylindrical body 51 has an inner diameter which is the same over theentire length thereof, and a surface formed by a flat portion 51a whichis parallel to the inner surface of the body, and a tapered portion 51bwhich tapers from the flat portion 51a to the end of the body. Anannular projection 52 is formed at the boundary of the flat portion 51aand tapered portion 51b. The second cylindrical body 53 has an outerdiameter which is the same over its entire length and comprises an endportion 53a having an inner diameter which grows successively larger asthe outer side of the end portion is approached and which is greaterthan the outer diameter of the flat portion 51a of cylindrical body 51,a tapered portion 53b having an inner surface corresponding to thesurface of the taper portion 51b of cylindrical body 51, and a flatportion 53c having an inner diameter equal to that of the cylindricalbody 51. Formed in the inner surface of the cyllindrical body 53 is anannular groove 54 corresponding to the annular projection 52 of thecylindrical body 51. When the cylindrical body 51 is pressed intocylindrical body 53 in the direction of the arrow, the cylindrical body53 spreads slightly at its end portion 53a to receive the cylindricalbody 51, the annular projection 52 whereof mates with the annular groove54 of the cylindrical body 53 to lock the bodies 51, 53 together.

EXAMPLE 1

Human plasma was mixed with sodium chloride and glycine and was leftstanding for 16 hr, followed by filtering the precipitate that formed.The amount of sodium chloride used was 30 g/dl plasma, and the amount ofglycine added (g/dl) was varied with respect to the amount of sodiumchloride. The total amount of protein (TP) and the amounts of albumin(Alb) and globulins (Glo) contained in the plasma before and aftertreatment are shown in Table 1. The amounts of immunoglobulins (Ig)G,IgA and IgM contained in the globulins are also shown in the table.

                  TABLE 1                                                         ______________________________________                                                                             IgA  IgM                                         T P   Alb     Glo     IgG    (mg/ (mg/                                        (g/dl)                                                                              (g/dl)  (g/dl)  (mg/dl)                                                                              dl)  dl)                                 ______________________________________                                        BEFORE    6.4     3.8     2.6   1270   216  134                               PROCESSING                                                                    AMOUNT   0    5.7     3.5   2.2   1180   196  124                             OF       5    4.8     3.2   1.6    680   136   96                             GLYCINE 10    4.2     3.0   1.2    380   100   90                             ADDED   15    3.8     2.9   0.9    240    72   66                                     20    3.3     2.5   0.8    90     38   60                             ______________________________________                                    

The tabulated results show that mixing glycine with sodium chloridegreatly enhances the effectiveness of selective precipitative separationof macromolecular protein from plasma.

EXAMPLE 2

Human plasma was treated by using the methodology of Example 1 with amixture of 30 g/dl (plasma) of sodium chloride and 8 g/dl (plasma) ofcystine, 8 g/dl (plasma) of tyrosine or 8 g/dl (plasma) ofN-acetyltryptophan. The total amount of protein, the amounts of albuminand globulin, and the ratio (A/G) of albumin to globulin are shown inTable 2. The results of adding glycine in the same concentration ascystine, tyrosine or N-acetyltryptophan are also shown.

                  TABLE 2                                                         ______________________________________                                                      T P   Alb     Glo                                                             (g/dl)                                                                              (g/dl)  (g/dl)  A/G                                       ______________________________________                                        BEFORE          5.8     2.5     3.3   0.76                                    PROCESSING                                                                    AMINO  NONE         4.0     2.1   1.9   1.11                                  ACID   CYSTINE      3.9     2.1   1.8   1.17                                  ADDED  TYROCINE     3.8     2.0   1.7   1.17                                         GLYCINE      3.0     2.0   1.0   2.00                                         N--ACETYL    3.1     1.8   1.3   1.38                                         TRYPTOPHAN                                                             ______________________________________                                    

It will be appreciated from Table 2 that glycine is particularlyeffective among the amino acids used in the present invention.

As set forth above, the agent for the precipitative separation ofproteins from blood plasma according to the present invention enablesmacromolecular proteins, e.g., immunoglobulins and fibrinogen, to beefficiently removed in large quantity with a high degree of selectivity.When a chloride is used as the alkaline metal salt, even in highconcentration, large quantities of such useful proteins as albumin donot precipitate. This is particularly advantageous because it eliminatesthe need for strict control of concentration. In addition, among thealkaline metal chlorides, use of sodium chloride or potassium chlorideis particularly safe since these compounds are present in body fluid.

Further, though the apparatus for separating proteins from blood plasmaaccording to the present invention is simple in construction, theselective precipitative separation of macromolecular protein is possibleowing to use of the above-described separating agent. Moreover, by usingof a dialyzer for removing the plasma-protein separating agent from theplasma, the effectiveness of plasma purification is enhanced evenfurther since toxic substances having a molecular weight of from severalthousand to tens of thousands are removed simultaneously.

It is obvious that if the agent for the precipitative separation ofproteins from plasma according to the present invention is used as thesolution of the precipitating medium employed in the separatingapparatus of FIG. 1 or FIG. 2, the precipitative separating effect canbe greatly enhanced.

As many apparently widely different embodiments of the present inventioncan be made without departing from the spirit and scope thereof, it isto be understood that the invention is not limited to the specificembodiments thereof except as defined in the appended claims.

What we claim is:
 1. An apparatus for separation and removal of proteins from blood plasma, comprising:a vessel containing an agent for the precipitative separation of proteins from blood plasma, said agent comprising a mixture of an alkaline metal salt and an amino acid in an amount sufficient to promote a fractional precipitating effect of the alkaline metal salt with respect to plasma protein, said amino acid being selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acetyltryptophan, tyrosine and mixtures thereof; means for introducing plasma into said vessel; means for separating and removing a precipitate formed inside said vessel by bringing the separating agent and the plasma into contact therein; and means for removing at least some of the separating agent constituent from a plasma component which remains following removal of the precipitate.
 2. An apparatus for separaton and removal of proteins from blood plasma, comprising:a plasma pump for delivering plasma at a predetermined delivery rate, and for returning treated plasma in an amount equivalent to that of the plasma delivered; a vessel containing an agent for the precipitative separation of proteins from blood plasma, said agent comprising a mixture of an alkaline metal salt and an amino acid in an amount sufficient to promote a fractional precipitating effect of the alkaline metal salt with respect to plasma protein, said amino acid being selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acetyltryptophan, tyrosine and mixtures thereof; a precipitating medium pump for delivering said agent; a mixing unit for mixing the delivered plasma and the delivered agent, whereby a precipitate is formed in said mixing unit; a plasma filter for removing the precipitate formed in said mixing unit, thereby resulting in filtered plasma; a plasma constituent adjusting unit for removing the agent from the filtered plasma and for producing said treated plasma by adjusting the filtered plasma in terms of water content and electrolyte; and a control means for driving and controlling said plasma pump and said precipitating medium pump so that said precipitating medium pump is operatively associated with said plasma pump to deliver said agent at more than the predetermined delivery rate of said plasma pump.
 3. The apparatus according to claim 2, wherein said plasma constituent adjusting unit comprises:a further plasma pump operatively associated with said plasma pump for operating at a flow rate equal to or less than a delivery flow rate of said plasma pump; plasma concentrating means operatively associated with said further plasma pump for removing water from the plasma in an amount equal to or more than the amount of the agent mixed therewith, thereby resulting in concentrated plasma; and precipitating medium removing means for removing the agent from the concentrated plasma and for adjusting the electrolyte of the concentrated plasma.
 4. The apparatus according to claim 2, wherein said alkaline metal salt is a sodium chloride.
 5. The apparatus according to claim 2, wherein said neutral amino acid is a glycine.
 6. An apparatus for separation and removal of proteins from blood plasma, comprising:a main conduit section having a blood inlet means for receiving blood and a blood outlet means for supplying blood; a plasma separator and a plasma-blood mixing unit interposed in fluid communication in said main conduit section between said blood inlet means and said blood outlet means, said plasma separator separating plasma from blood introdcued from the blood inlet; a plasma pump for delivering the plasma separated by said plasma separator into a secondary conduit at a predetermined delivery rate, and for delivering treated plasma to said plasma-blood mixing unit in an amount equivalent to that of the plasma delivered into the secondary circuit; a vessel containing an agent for the precipitative separation of proteins from blood plasma, said agent comprising a mixture of an alkaline metal salt and an amino acid in an amount sufficient to promote a fractional precipitating effect of the alkaline metal salt with respect to plasma protein, said amino acid being selected from the group consisting of neutral amino acid, aspartic acid, cystine, N-acetyltryptophan, tyrosine and mixtures thereof; a precipitating medium pump for delivering said agent; a mixing unit for mixing the delivered plasma and the delivered agent, whereby a precipitate is formed in said mixing unit; a plasma filter for removing the precipitate formed in said mixing unit, thereby resulting in filtered plasma; a plasma constituent adjusting unit for removing the agent from the filtered plasma and for producing said treated plasma by adjusting the filtered plasma in terms of water content and electrolyte; and a control means for driving and controlling said plasma pump and said precipitating medium pump so that said precipitating medium pump is operatively associated with said plasma pump to deliver said agent at more than the predetermined delivery rate of said plasma pump. 